Maxwell® CSC Rapid ccfDNA Kit

High-yield and high-quality ccfDNA extracted from plasma collected in different blood collection tubes. Plasma was isolated from human whole blood collected in either Streck Cell-Free DNA BCT® (Streck plasma) or Vacutainer® K₂EDTA tubes (K₂EDTA plasma). ccfDNA was extracted from 4ml of plasma using either the Maxwell® CSC Rapid ccfDNA Kit or a manual competitor kit (Competitor Q) and eluted using 50µl of kit-specific Elution Buffer. ccfDNA yields were determined using either a fluorometric assay with a dsDNA-specific dye or a qPCR assay for amplification of a 75bp DNA target. Yields of ccfDNA extracted using the Maxwell® CSC Rapid ccfDNA Kit or the competitor kit are shown for Streck plasma (Panel A) and K₂EDTA plasma (Panel B). Data represent mean ±SD, N=4. ccfDNA yields produced by the Maxwell® CSC Rapid ccfDNA Kit were comparable to or better than those from Competitor Q, except for slightly higher yields from K₂EDTA plasma observed with Competitor Q in the qPCR assay, possibly due to contaminating genomic DNA. To assess ccfDNA quality, purified ccfDNA samples were analyzed by Agilent Cell-Free DNA ScreenTape Assay. Panel C. Representative TapeStation electropherograms of the same ccfDNA samples extracted from Streck plasma shown in Panel A. Panel D. Representative TapeStation electropherograms of the same ccfDNA samples extracted from K₂EDTA plasma shown in Panel B. The TapeStation electropherogram for Competitor Q in Panel D shows a higher concentration of contaminating genomic DNA in ccfDNA extracted from K₂EDTA plasma (indicated by red circle) as compared to the ccfDNA extracted using the Maxwell® CSC Rapid ccfDNA Kit. A large amount of genomic DNA carryover in ccfDNA extracted from K₂EDTA plasma by Competitor Q resulted in higher reported yield of ccfDNA in the qPCR assay in Panel B, confounding the actual ccfDNA yield.

ccfDNA compatible with next-generation sequencing. ccfDNA was extracted from 4ml of plasma isolated from Streck Cell-Free DNA BCT® using either the Maxwell® CSC Rapid ccfDNA Kit or a competitor ccfDNA extraction kit (Competitor Q). Purified ccfDNA samples were analyzed in a Whole Genome Sequencing assay. Successful libraries were generated for all the samples, and sequencing was performed on a NovaSeq X Plus system (Illumina), using 2 × 150 paired-end sequencing in a shared-lane configuration, with 140–170M reads per library and subsampling of 70M reads per sample. Panel A. A GC-bias plot was generated for ccfDNA extracted with the Maxwell® CSC Rapid ccfDNA Kit (yellow line) and Competitor Q (blue line) to determine how evenly AT- and GC-rich regions are covered using Picard CollectGcBiasMetrics tool (MultiQC: Summarized analysis results for multiple tools and samples in a single report. Philip Ewels, Måns Magnusson, Sverker Lundin and Max Käller Bioinformatics (2016) doi: 10.1093/bioinformatics/btw354. PMID: 27312411). The curve for ccfDNA extracted using the Maxwell® CSC Rapid ccfDNA Kit tracks the ideal flat line (1.0) more closely at low and high GC, indicating lower bias and, therefore, more uniform coverage across the genome as compared to ccfDNA extracted with the competitor kit. Panel B. Sequencing metrics of the same ccfDNA samples presented in Panel A. The sequencing results demonstrated comparable performance between the Maxwell® CSC Rapid ccfDNA Kit and the competitor kit.